Sequence-specific hybridization of oligonucleotide probes is a useful and very valuable reaction for detecting and identifying a specific polynucleotide sequence. This identification of a specific oligonucleotide sequence requires a readout system that produces a signal indicating hybridization of the specific target sequence to an oligonucleotide probe. A popular readout system is fluorescent labeling of a DNA probe, which creates a fluorescent signal in response to a specific hybridization reaction. Fluorescence labeled probes and sequence-specific methods of their use generally employ a soluble water phase nucleic acid that is labeled with a reporter moiety such as a fluorescent label, to facilitate detection of probe hybridization. Some of these methods employ fluorescence energy transfer ("FRET") to detect probe hybridization rather than direct detection of fluorescence intensity.
In the FRET technique a light source illuminates the sample. Energy of an absorbed photon from the light source can transfer from a donor fluorophore to an acceptor dye (which may or may not be a fluorophore) when (i) the absorption spectrum of the acceptor dye overlaps the emission spectrum of the excited fluorophore and (ii) the two molecules are in close proximity. The excited-state energy of the donor fluorophore transfers to the neighboring acceptor by the phenomenon of resonance dipole-induced dipole interaction, thereby causing quenching of the donor fluorescence. Alternatively, if the acceptor also is a fluorophore, the intensity of its fluorescence may be enhanced. The efficiency of energy transfer is highly dependent on the distance between the donor and acceptor, and equations predicting these relationships have been developed by Forster (Ann. Phys. 2:55-75 (1948)). The distance between donor and acceptor dyes at which energy transfer efficiency is 50% is referred to as the Forster distance (R[o]). Other mechanisms of fluorescence quenching also are known including, for example, charge transfer and collisional quenching.
The FRET technique is particularly useful for detecting hybridization of nucleic acid because of a marked change in the fluorescence properties of donor fluor and/or acceptor dye label when they are brought in close physical proximity by the hybridization of two complementary oligonucleotides. In this format, this change in fluorescence may be measured as a change in the amount of energy transfer or as a change in the amount of fluorescence quenching, and typically is indicated as an increase in the fluorescence intensity of one of the dyes. Thus, the FRET technique potentially can distinguish between unhybridized and hybridized oligonucleotide species without the need to physically separate the species.
Simple FRET systems rely on hybridization between two separate complementary oligonucleotides, one labeled with the donor fluorophore and one labeled with the acceptor. When hybridization occurs, leading to a double-stranded oligonucleotide, quenching and/or increased energy transfer leads to a decrease in donor fluorescence as compared to the fluorescence from the individual single-stranded oligonucleotides. Several formats for FRET hybridization assays are reviewed in Nonisotopic DNA Probe Techniques (1992. Academic Press, Inc., pgs. 311-352) and in WO 97/22719.
Alternatively, the donor and acceptor may be linked to a single oligonucleotide and used to monitor a change between a hairpin conformation and a non-hairpin conformation of the oligonucleotide. In this format, donor fluorescence decreases when an internal hairpin structure is formed and increases when the hairpin dissociates, for example when a complementary region of the oligonucleotide hybridizes with a separate oligomer instead of itself. For example, a partially self-complementary oligonucleotide may be dye-labeled at both ends and may form a hairpin between the ends, bringing the two dyes into close proximity and permitting energy transfer and quenching between the dyes. Hybridization of an internal region of the oligomer with a second nucleic acid disrupts the hairpin and increases the distance between the two dyes, thus reducing quenching, and allowing fluorescent dyes to emit photons upon their excitation.
The FRET technique has been used to detect a change of a hairpin structure in solution as described by U.S. Pat. No. 5,332,659, issued Jul. 26, 1994 and by WO 97/22719, published Jun. 26, 1997. The FRET technique disclosed in those publications does not require a separation step to measure hybridization and furthermore can use reagents that are more stable than alternative reagents such as radioisotopic or enzyme labeled probes. Unfortunately, the disclosed FRET techniques cannot be used to simultaneously observe hybridization events of multiple targets, particularly when the target molecules are present in the same solution. This is a serious deficiency for modern genetic analyses which require the ability to use multiple DNA probes to determine the presence of multiple sequences in a single sample.
Another problem in the art is that a fluorescently labeled nucleic acid reagent, such as those described above, cannot easily be reused. Yet another problem is that the labeled nucleic acid must contain a second dye that quenches the first. Dyes typically are hydrophobic, which leads to self-association in aqueous solution, with consequent steric hindrance of base pair formation. Such steric hindrance is a significant barrier to duplex formation when the oligonucleotide is small and the two dyes have a correspondingly larger steric effect.
Yet another problem is that the solution methods use a detection system that relies on a difference in wavelength of emission (U.S. Pat. No. 5,332,659) or intensity (WO 97/22719, WO 97/39008) that is superimposed on a much higher light emission background. The high light emission background significantly limits detection sensitivity. Furthermore, only a few probes can be used and even these suffer great loss of sensitivity because the excitation and emission light from one probe species contributes to the background when measuring another species. Accordingly, a great need exists for an assay device and method that can take advantage of the sensitivity of fluorescence detection but which does not suffer from the problems described above.